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OPEN
Received: 16 February 2017 Accepted: 25 April 2017 Published: xx xx xxxx
Chlorophyll derivatives enhance invertebrate red-light and ultraviolet phototaxis Andrea Degl’Innocenti1, Leonardo Rossi 2, Alessandra Salvetti2, Attilio Marino3, Gabriella Meloni1,4, Barbara Mazzolai1 & Gianni Ciofani 3,5 Chlorophyll derivatives are known to enhance vision in vertebrates. They are thought to bind visual pigments (i.e., opsins apoproteins bound to retinal chromophores) directly within the retina. Consistent with previous findings in vertebrates, here we show that chlorin e6 — a chlorophyll derivative — enhances photophobicity in a flatworm (Dugesia japonica), specifically when exposed to UV radiation (λ = 405 nm) or red light (λ = 660 nm). This is the first report of chlorophyll derivatives acting as modulators of invertebrate phototaxis, and in general the first account demonstrating that they can artificially alter animal response to light at a behavioral level. Our findings show that the interaction between chlorophyll derivatives and opsins virtually concerns the vast majority of bilaterian animals, and also occurs in visual systems based on rhabdomeric (rather than ciliary) opsins. In order to sense light, animals rely on specialized macromolecules, namely photoreceptor proteins; these are required for the initial sensory transduction event that eventually leads to vision. At least two groups of photoreceptor proteins are known so far, and a third one has recently been proposed1. The most diversified and widely distributed type of photoreceptor protein is opsins. Opsins belong to a multifamily of membrane proteins known as G protein-coupled receptors (GPCRs), or seven-transmembrane receptors. GPCR genes are extremely abundant — among others — in animal and fungal genomes, and GPCRs constitute the most common target for drugs, cf., ref. 2. Opsins bind retinal, a chromophore that captures light and transfers energy to the apoprotein; together they form a visual pigment. Photons within a certain stretch of wavelengths are trapped: absorption range ultimately depends on the structure of retinal, as well as on the way this contacts its binding pocket within the opsin (reviewed in ref. 3). However, there are a few instances where the spectral tuning of an opsin does not depend solely on these factors. In fact, the meso-bathypelagic fish Malacosteus niger is capable of seeing far-red light in spite of the absorption spectrum of its opsins, which are green-tuned and display negligible absorption already at λ = 650 nm. Through its diet, the fish concentrates chlorophyll derivatives (CDs) within its retina, in the same anatomic compartment that contains visual pigments, the rod outer segment (ROS). Specifically, M. niger accumulates demetallated and defarnesylated derivatives of bacteriochlorophylls C and D4–8. Following this natural example, some studies explored the possibility of CDs being modulators of vertebrate vision. A handful of CDs were tested as candidate photosensitizers of purified bovine rhodopsin (an opsin): one of them, a substituted chlorin named chlorin e6 (Ce6, Fig. 1), proved to be a potent bleaching agent for the protein upon exposure to red light9. Together with metal ions, it could also stabilize rhodopsin against thermal denaturation2. Another study showed that living rod cells, extracted from the salamander Ambystoma tigrinum, respond better to red light if exposed to Ce610. When injected intravenously in a mouse model Ce6 could rapidly accumulate in the ROS, and caused improved electroretinogram responses to red (>640 nm) and blue (456 ± 30 nm) wavelengths11. Intravenous administration of Ce6 (or related compounds) in macaque and rabbit also led to 1 Center for Micro-BioRobotics, Istituto Italiano di Tecnologia, Viale Rinaldo Piaggio 34, 56025, Pontedera (Pisa), Italy. 2Department of Clinical and Experimental Medicine, Università di Pisa, Via Alessandro Volta 4, 56126, Pisa, Italy. 3Smart Bio-Interfaces, Istituto Italiano di Tecnologia, Viale Rinaldo Piaggio 34, 56025, Pontedera (Pisa), Italy. 4 The BioRobotics Institute, Scuola Superiore Sant’Anna, Viale Rinaldo Piaggio 34, 56025, Pontedera (Pisa), Italy. 5 Department of Mechanical and Aerospace Engineering, Politecnico di Torino, Corso Duca degli Abruzzi 24, 10129, Torino, Italy. Correspondence and requests for materials should be addressed to A.D. (email: andrea.deglinnocenti@ iit.it) or G.C. (email: [email protected])
Scientific Reports | 7: 3374 | DOI:10.1038/s41598-017-03247-1
1
www.nature.com/scientificreports/
Figure 1. Chlorin e6 enhances UV and red-light avoidance in Dugesia japonica. (A) Skeletal formula of chlorin e6. (B) Absorption and emission (smaller graph, excitation wavelength ~405 nm) spectra of chlorin e6 dissolved in 1% v/v dimethyl sulfoxide; colored boxes approximate wavelength positions (exact values in gray, nm) for the tested colors (left to right: UV, cyan, red, NIR). (C) Representation of the experimental chamber used for behavioral experiments, with LED light diffusing from the right edge; animals found within the darkest quadrant (area delimited by the vertical dashed line) after two minutes of light exposure were counted as photophobic. (D) Bar charts reporting the percentage of photophobic animals, either treated with chlorin e6 (C) or plain dimethyl sulfoxide (D), after exposure to light of different wavelengths (charts from left to right: UV, cyan, red, NIR) at different radiant power. Grey bars illustrate control experiments with no light stimulus provided. Grey asterisks indicate significance for unpaired one-tailed T-test; black asterisks indicate significance for unpaired one-tailed T-test and two-tailed Mann Whitney U-test. The number of asterisks specifies significance for different p-value threshold of the unpaired one-tailed T-test (*for p
OPEN
Received: 16 February 2017 Accepted: 25 April 2017 Published: xx xx xxxx
Chlorophyll derivatives enhance invertebrate red-light and ultraviolet phototaxis Andrea Degl’Innocenti1, Leonardo Rossi 2, Alessandra Salvetti2, Attilio Marino3, Gabriella Meloni1,4, Barbara Mazzolai1 & Gianni Ciofani 3,5 Chlorophyll derivatives are known to enhance vision in vertebrates. They are thought to bind visual pigments (i.e., opsins apoproteins bound to retinal chromophores) directly within the retina. Consistent with previous findings in vertebrates, here we show that chlorin e6 — a chlorophyll derivative — enhances photophobicity in a flatworm (Dugesia japonica), specifically when exposed to UV radiation (λ = 405 nm) or red light (λ = 660 nm). This is the first report of chlorophyll derivatives acting as modulators of invertebrate phototaxis, and in general the first account demonstrating that they can artificially alter animal response to light at a behavioral level. Our findings show that the interaction between chlorophyll derivatives and opsins virtually concerns the vast majority of bilaterian animals, and also occurs in visual systems based on rhabdomeric (rather than ciliary) opsins. In order to sense light, animals rely on specialized macromolecules, namely photoreceptor proteins; these are required for the initial sensory transduction event that eventually leads to vision. At least two groups of photoreceptor proteins are known so far, and a third one has recently been proposed1. The most diversified and widely distributed type of photoreceptor protein is opsins. Opsins belong to a multifamily of membrane proteins known as G protein-coupled receptors (GPCRs), or seven-transmembrane receptors. GPCR genes are extremely abundant — among others — in animal and fungal genomes, and GPCRs constitute the most common target for drugs, cf., ref. 2. Opsins bind retinal, a chromophore that captures light and transfers energy to the apoprotein; together they form a visual pigment. Photons within a certain stretch of wavelengths are trapped: absorption range ultimately depends on the structure of retinal, as well as on the way this contacts its binding pocket within the opsin (reviewed in ref. 3). However, there are a few instances where the spectral tuning of an opsin does not depend solely on these factors. In fact, the meso-bathypelagic fish Malacosteus niger is capable of seeing far-red light in spite of the absorption spectrum of its opsins, which are green-tuned and display negligible absorption already at λ = 650 nm. Through its diet, the fish concentrates chlorophyll derivatives (CDs) within its retina, in the same anatomic compartment that contains visual pigments, the rod outer segment (ROS). Specifically, M. niger accumulates demetallated and defarnesylated derivatives of bacteriochlorophylls C and D4–8. Following this natural example, some studies explored the possibility of CDs being modulators of vertebrate vision. A handful of CDs were tested as candidate photosensitizers of purified bovine rhodopsin (an opsin): one of them, a substituted chlorin named chlorin e6 (Ce6, Fig. 1), proved to be a potent bleaching agent for the protein upon exposure to red light9. Together with metal ions, it could also stabilize rhodopsin against thermal denaturation2. Another study showed that living rod cells, extracted from the salamander Ambystoma tigrinum, respond better to red light if exposed to Ce610. When injected intravenously in a mouse model Ce6 could rapidly accumulate in the ROS, and caused improved electroretinogram responses to red (>640 nm) and blue (456 ± 30 nm) wavelengths11. Intravenous administration of Ce6 (or related compounds) in macaque and rabbit also led to 1 Center for Micro-BioRobotics, Istituto Italiano di Tecnologia, Viale Rinaldo Piaggio 34, 56025, Pontedera (Pisa), Italy. 2Department of Clinical and Experimental Medicine, Università di Pisa, Via Alessandro Volta 4, 56126, Pisa, Italy. 3Smart Bio-Interfaces, Istituto Italiano di Tecnologia, Viale Rinaldo Piaggio 34, 56025, Pontedera (Pisa), Italy. 4 The BioRobotics Institute, Scuola Superiore Sant’Anna, Viale Rinaldo Piaggio 34, 56025, Pontedera (Pisa), Italy. 5 Department of Mechanical and Aerospace Engineering, Politecnico di Torino, Corso Duca degli Abruzzi 24, 10129, Torino, Italy. Correspondence and requests for materials should be addressed to A.D. (email: andrea.deglinnocenti@ iit.it) or G.C. (email: [email protected])
Scientific Reports | 7: 3374 | DOI:10.1038/s41598-017-03247-1
1
www.nature.com/scientificreports/
Figure 1. Chlorin e6 enhances UV and red-light avoidance in Dugesia japonica. (A) Skeletal formula of chlorin e6. (B) Absorption and emission (smaller graph, excitation wavelength ~405 nm) spectra of chlorin e6 dissolved in 1% v/v dimethyl sulfoxide; colored boxes approximate wavelength positions (exact values in gray, nm) for the tested colors (left to right: UV, cyan, red, NIR). (C) Representation of the experimental chamber used for behavioral experiments, with LED light diffusing from the right edge; animals found within the darkest quadrant (area delimited by the vertical dashed line) after two minutes of light exposure were counted as photophobic. (D) Bar charts reporting the percentage of photophobic animals, either treated with chlorin e6 (C) or plain dimethyl sulfoxide (D), after exposure to light of different wavelengths (charts from left to right: UV, cyan, red, NIR) at different radiant power. Grey bars illustrate control experiments with no light stimulus provided. Grey asterisks indicate significance for unpaired one-tailed T-test; black asterisks indicate significance for unpaired one-tailed T-test and two-tailed Mann Whitney U-test. The number of asterisks specifies significance for different p-value threshold of the unpaired one-tailed T-test (*for p
